1. Field of the Invention
The current application relates to a confocal laser microscope with at least one laser whose illumination light is transmitted in direction of the microscope objective by at least one light-conducting fiber.
2. Description of Related Art
Laser scanning systems use lasers of different power classes. Further, a laser scanning system is characterized by a large quantity of variable modules serving as detectors or for illumination.
A confocal scanning microscope contains a laser module which preferably has a plurality of laser beam sources generating illumination light of different wavelengths. A scanning device into which the illumination light is coupled as illumination beam has a main color splitter, an X-Y scanner, and a scanning objective for guiding the illumination beam by beam deflection over a sample located on a microscope stage of a microscope unit. A measurement light beam which is generated in this way coming from the sample is directed to at least one confocal detection diaphragm (detection pinhole) of a detection channel by a main color splitter and imaging optics.
FIG. 1 is schematic diagram showing this type of beam path of a laser scanning microscope. As is shown, the modules are a light source, scan module, detection unit, and microscope. These modules are described in more detail in the following. In addition, reference is had to DE19702753A1.
Lasers of different wavelengths are used in a LSM for specific excitation of the different dyes in a specimen. The choice of excitation wavelength is governed by the absorption characteristics of the dyes to be examined. The excitation beam is generated in the light source module. Different lasers (argon, argon-krypton, TiSa) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength are carried out in the light source module, e.g., by means of an acousto-optical crystal. Subsequently, the laser radiation reaches the scan module through a fiber or a suitable mirror arrangement.
The laser radiation generated in the light source is focused in the specimen in a diffraction-limited manner by means of the objective via the scanner, the scan optics and the tube lens. The focus scans the specimen point by point in the X-Y direction. The pixel dwell times during the scan over the sample are usually in the range of less than one microsecond to several hundred microseconds.
In case of a confocal detection (descanned detection) of fluorescent light, the light which is emitted from the focus plane (specimen) and from the planes above and below the latter travels to a dichroic beamsplitter (MD) by way of the scanner. This dichroic beamsplitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole) which is located exactly in a plane conjugate to the focus plane. Fluorescent light located outside the focus is suppressed in this way. The optical resolution of the microscope can be adjusted by varying the aperture size. Another dichroic blocking filter (EF) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by a point detector (PMT).
When multiphoton absorption is used, the excitation of the dye fluorescence takes place within a small volume in which the excitation intensity is especially high. This area is only negligibly larger than the detected area when using a confocal arrangement. Therefore, a confocal diaphragm can be dispensed with and detection can be carried out directly after the objective (non-descanned detection).
In another arrangement for detection of a dye fluorescence excited by multiphoton absorption, descanned detection is carried out, but this time the pupil of the objective is imaged in the detection unit (non-confocal descanned detection).
In a three-dimensionally illuminated image, both detection arrangements in connection with the corresponding single-photon or multiphoton absorption will display only the plane (optical section) located in the focus plane of the objective. A three-dimensional image of the sample can then be generated with the help of a computer by recording a plurality of optical sections in the X-Y plane at different depths Z of the sample. Accordingly, the LSM is suitable for examining thick specimens. The excitation wavelengths are determined by the dye employed with its specific absorption characteristics. Dichroic filters suited to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Currently, in biomedical applications a plurality of different cell regions are labeled simultaneously by different dyes (multiflourescence). In the prior art, the individual dyes can be detected separately based either on different absorption characteristics or on emission characteristics (spectra). For this purpose, an additional splitting of the fluorescent light of a plurality of dyes is carried out by the secondary beamsplitters (DBS) and the individual dye emissions are detected separately in separate point detectors (PMT x).
A very fast line scanner with image generation at 120 images per second is realized in the LSM LIVE by Carl Zeiss MicroImaging GmbH. (http://www.zeiss.de/c12567be00459794/Contents-Frame/fd9a0090eee01a641256a550036267b).
As a rule, the light source modules are connected to the scan module by light-conducting fibers.
It is known from DE19702753A1 to provide displaceable collimating optics for coupling the laser light from the light guide outputs into the microscope beam path. For example, a movable collimator which compensates for the longitudinal chromatic aberration of the objective being used is arranged behind the plug-in glass fiber input coupling of the UV (or 405 nm) illumination so that the focus points of UV and visible light again lie in a plane. The movable collimator is moved into another position for every objective.
Static collimating lenses in the fiber-optic plug are mentioned in DE10361176 A1.
For purposes of transporting, when replacing defective fibers, and possibly for coupling in other lasers, there is a need for a glass fiber input coupling which can be unplugged and then plugged in again without adjustment.
DE 19829988 discloses an adjustment of fibers in more than one spatial direction, but this is relatively complicated.
There are technical problems which arise from the demand for fibers which do not require adjustment: A standard single mode glass fiber, e.g., for the wavelength of 405 nm, typically has a mode field diameter of 3.5 μm and a numerical aperture of 0.1. For purposes of plugging in in a reproducible manner without requiring adjustment, this means that this fiber must be positioned with a positioning accuracy of appreciably less than 1 μm for repeatedly plugging in at the scanning head, while the angular accuracy need only be in the range of 10 mrad. To ensure the lateral superposition of different wavelengths which are directly coupled into the scanning head separately by means of fibers and to ensure the positioning accuracy of the laser beam with respect to the system pupil, the reproducibility of a simple glass fiber plug-in connection must be appreciably better than 1 μm, which realistically cannot be implemented at a reasonable expenditure on mechanical adjustment.